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The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

Timmerman SL, Pfingsten JS, Kieft JS, Krushel LA - PLoS ONE (2007)

Bottom Line: Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active.However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1).Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado, USA.

ABSTRACT
A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.

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The mouse TrkB 5′ leaders exhibit IRES activity when expressed in dicistronic RNA constructs.Dicistronic luciferase mRNA containing the β-globin and the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio for each construct was normalized to that of the negative control, β-globin.
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pone-0003242-g004: The mouse TrkB 5′ leaders exhibit IRES activity when expressed in dicistronic RNA constructs.Dicistronic luciferase mRNA containing the β-globin and the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio for each construct was normalized to that of the negative control, β-globin.

Mentions: To overcome the limitations of cryptic promoter activity (and cryptic splicing which we did not examine), we in vitro transcribed the dicistronic DNA. The resulting dicistronic mRNA was transfected into C6 cells. All three TrkB 5′ leaders exhibited a P∶R ratio higher than that observed from the dicistronic mRNA containing the β-globin 5′ leader (Fig. 4). The largest ratio of approximately six was seen with L2. Ex2 generated a P∶R ratio of four, and L1 showed the lowest ratio of approximately 2.5. This result demonstrates that all three mouse TrkB 5′ leaders can mediate IRES-dependent translation. The result also indicates that the presence of a cryptic promoter in a 5′ leader does not preclude its ability to internally initiate translation.


The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

Timmerman SL, Pfingsten JS, Kieft JS, Krushel LA - PLoS ONE (2007)

The mouse TrkB 5′ leaders exhibit IRES activity when expressed in dicistronic RNA constructs.Dicistronic luciferase mRNA containing the β-globin and the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio for each construct was normalized to that of the negative control, β-globin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2531235&req=5

pone-0003242-g004: The mouse TrkB 5′ leaders exhibit IRES activity when expressed in dicistronic RNA constructs.Dicistronic luciferase mRNA containing the β-globin and the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio for each construct was normalized to that of the negative control, β-globin.
Mentions: To overcome the limitations of cryptic promoter activity (and cryptic splicing which we did not examine), we in vitro transcribed the dicistronic DNA. The resulting dicistronic mRNA was transfected into C6 cells. All three TrkB 5′ leaders exhibited a P∶R ratio higher than that observed from the dicistronic mRNA containing the β-globin 5′ leader (Fig. 4). The largest ratio of approximately six was seen with L2. Ex2 generated a P∶R ratio of four, and L1 showed the lowest ratio of approximately 2.5. This result demonstrates that all three mouse TrkB 5′ leaders can mediate IRES-dependent translation. The result also indicates that the presence of a cryptic promoter in a 5′ leader does not preclude its ability to internally initiate translation.

Bottom Line: Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active.However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1).Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado, USA.

ABSTRACT
A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.

Show MeSH